We have developed a model for studying tolerance to persistent low dose antigen in vivo, which results in the generation of a large number of anergic (hyporesponsive) T cells. We call this state adaptive tolerance. We inject CD4+, antigen-specific T cells from a T cell receptor transgenic mouse on a Rag2-/- background (a monospecific T cell population) into a second transgenic mouse expressing the antigen in a lymphopenic background (no other T cells). Within 24 hours after transfer, the T cells are all activated by the antigen (as evidenced by an increase in size and expression of CD69), and proliferate extensively for several days, increasing in number about 100-fold. This expansion is followed by a deletional phase during which 50% of the cells disappear. Finally, the population reaches a steady state level in which the cells appear to be refractory to restimulation in vivo and in vitro. In this adaptive tolerant state, cytokine responses to high doses of antigen in vitro are inhibited 90%. However, in vivo bromodeoxyuridine labeling shows a slow T cell turnover of about 5% of the cells per day, and B cell help is still sufficient for the mice to eventually develop a mild form of chronic autoimmune arthritis or polychondritis depending on the antigen and the T cell specificity. The hyporesponsive state is reversible if the cells are transferred again into a second host not expressing the antigen. If the retransfer is into a host expressing the antigen, the T cells remain hyporesponsive and slowly decrease their IL-2 and IFN gamma production by another 6-10 fold over 3-4 weeks. This deeper state of anergy suggests that the tolerance process is adaptable to different levels. Finally, if the initial antigen-bearing host is T cell replete, i.e., contains a normal polyclonal T cell population, then the naive T cell response is blunted. The expansion phase is curtailed by 5 to 10 fold, the differentiation into effector T cells (producing IFN-gamma) does not occur, and the anergic T cells disappear with a half life of 9-12 days. As a consequence, the mice do not develop any autoimmune disease. [unreadable] In FY 2008 the lab has focused on determining what causes the difference in the antigen-specific naive CD4+ T cell response when the cells are transferred into T cell-sufficient versus T cell-deficient antigen-bearing hosts. The difference must somehow be related to the presence of other T cells in the environment. The critical cells are peripheral alpha/beta T cells because adult thymectomy does not alter the full-host effects and introducing the TCR alpha KO mutation eliminates them all. To identify the inhibitory T cell we first depleted the T cell-sufficient, host of CD25+ regulatory T cells by pretreatment with both anti-CD25 and anti-IL-2 mAbs. This had no impact on the blunted expansion or shortened half-life of a subsequently transferred population of antigen-specific, naive T cells. We then reconstituted T cell-deficient mice with normal CD4+/CD25+, CD4+/CD25-, or CD8+ T cells purified by flow cytometry. One to 7 days later, naive antigen-specific CD4+ T cells were injected and their expansion and longevity monitored. Both expanded polyclonal CD4+ T cell populations partially facilitated the loss of the antigen-specific T cells after the latter became anergic. This decrease from peak cell numbers occurred over a 3 week time period and was about 20 fold, compared to only a 2 fold loss in a host reconstituted with CD8+ T cells. Both CD25- and CD25+ T cells produced this effect, although CD25- T cells appeared to be more efficient. Since these experiments suggested that CD4+CD25- T cells could mediate at least one of the effects in a full host, we considered the possibility that the differences in outcome in the T cell-sufficient host were caused by T cell competition for nutrients, growth factors, or activation niches. To test this type of model we transferred the original antigen-specific T cells into a second TCR transgenic, Rag2-/- host specific for another antigen, but also expressing the first antigen. These hosts contain around 5 to 10 million naive T cells filling the space in lymph nodes and spleen. Despite this, the original naive antigen-specific T cells adoptively transferred into each such host expanded fully (100-fold), differentiated into IFN-gamma producers, entered the adaptive tolerant state, and persisted for months. This result was observed with 5 different CD4+ TCR transgenics. One of them was specific for an antigen presented by the same MHC molecule as that used by the first antigen. Thus, non-specific competition for things such as IL-7 would seem not to be responsible for the inhibitory effects of the polyclonal T cell population. We are currently carrying out experiments to try and clone out the inhibitory CD4+ T cell population to further determine its phenotype and characterize its mechanism of inhibition.[unreadable] The second goal of the laboratory in FY 2008 was to understand the molecular mechanism(s) underlying T cell adaptive tolerance. Our earlier studies had pinpointed a block in T cell receptor signaling at the level of ZAP-70 phosphorylation of down-stream substrates such as the Linker of Activated T cells (LAT). These initial biochemical experiments were done by activating the T cells with monoclonal antibodies to TCR, CD4, and/or CD28. When we turned to confirming our observations using antigen presentation by APC, a defect in immunologic synapse formation was discovered in addition to the ZAP-70 phosphorylation defect. Although Rap1-GTP formation (required for inside out signaling) was somewhat diminished following anti-TCR and anti-CD4 stimulation, LFA-1 dependent conjugate formation was largely intact. This is because the APC used, the transfected fibroblast line P13.9, expresses large amounts of its ligand ICAM-1, and because the T cell membrane expression of LFA-1 is stably enhanced 3 fold in the tolerant T cells during the activation and effector cell differentiation phases that take place prior to the onset of adaptive tolerance. Despite this tight cell/cell interaction, however, translocation of ZAP-70 and the TCR to the contact region between the T cell and the APC was greatly impaired. Downstream signaling molecules such as LAT, PLC-gamma-1, and PKC-theta were also not concentrated there. Surprisingly, Vav-1 activation (by phosphorylation) was normal, even though actin polymerization at the synapse and microtubular organizing center (MTOC) reorientation toward the synapse were impaired. Phosphorylation of cdc42 by Vav, which is required for actin polymerization, was also normal. We suspect, however, that Vav and cdc42 are not being mobilized effectively to the synapse and thus preventing actin polymerization at that site from stabilizing the structure. The mechanism behind this failure is currently under investigation.